Effective molecular RHD typing strategy for blood donations

Abstract

Background: More than 50 weak D alleles and numerous partial D alleles have been described to date that can be identified by molecular methods as polymerase chain reaction (PCR) and DNA sequencing of the RHD gene. A real time-based RHD typing scheme was developed and tested during an 8-month period.

Study design and methods: A total of 53,347 blood donors and patients were tested with standardized immunohematologic methods. A total of 201 DNA samples with weak D reactions underwent molecular characterization by weak D real-time PCR, exon-screening real-time PCR, and nucleotide sequencing of RHD Exons 1 through 10. A total of 2,427 samples with D- phenotype were tested for the presence of RHD markers.

Results: Molecular typing of 201 samples with weak D expression revealed 15 different known aberrant alleles as well as one new weak D type dubbed weak D Type 49. Approximately 60 percent of the alleles were determined as weak D Types 1 through 3 and detected by only one amplification run. Weak D Type 1 represented the most frequent allele (n = 72). Three samples with D- phenotype showed amplification of RHD-specific markers. Sequence-based typing (SBT) of these samples revealed a DEL allele, RHD(IVS3+1G>A), in two samples and one weak D Type 4.3.

Conclusions: The presented scheme for RHD genotyping of weak D red blood cell units was reliable for detection of aberrant alleles. Testing of D- blood samples as quality control seems to overcome limitations of standard serology by detection of samples with weak D or DEL phenotype.